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Supplemental Data.Pdf Supplementary material -Table of content Supplementary Figures (Fig 1- Fig 6) Supplementary Tables (1-13) Lists of genes belonging to distinct biological processes identified by GREAT analyses to be significantly enriched with UBTF1/2-bound genes Supplementary Table 14 List of the common UBTF1/2 bound genes within +/- 2kb of their TSSs in NIH3T3 and HMECs. Supplementary Table 15 List of gene identified by microarray expression analysis to be differentially regulated following UBTF1/2 knockdown by siRNA Supplementary Table 16 List of UBTF1/2 binding regions overlapping with histone genes in NIH3T3 cells Supplementary Table 17 List of UBTF1/2 binding regions overlapping with histone genes in HMEC Supplementary Table 18 Sequences of short interfering RNA oligonucleotides Supplementary Table 19 qPCR primer sequences for qChIP experiments Supplementary Table 20 qPCR primer sequences for reverse transcription-qPCR Supplementary Table 21 Sequences of primers used in CHART-PCR Supplementary Methods Supplementary Fig 1. (A) ChIP-seq analysis of UBTF1/2 and Pol I (POLR1A) binding across mouse rDNA. UBTF1/2 is enriched at the enhancer and promoter regions and along the entire transcribed portions of rDNA with little if any enrichment in the intergenic spacer (IGS), which separates the rDNA repeats. This enrichment coincides with the distribution of the largest subunit of Pol I (POLR1A) across the rDNA. All sequencing reads were mapped to the published complete sequence of the mouse rDNA repeat (Gene bank accession number: BK000964). The graph represents the frequency of ribosomal sequences enriched in UBTF1/2 and Pol I-ChIPed DNA expressed as fold change over those of input genomic DNA. The lower panel represents a schematic of a single rDNA repeat. Scale bars in kb are shown below; the site of transcription initiation start is indicated at 0kb. En: enhancer elements; ETS: external transcribed spacer; IGS: intergenic spacer. (B) UBTF1/2 is localized to active NORs. Combined immunofluorescence (IF)-FISH analysis of UBTF1/2 and rDNA in interphasic human fibroblasts. Active NORs are associated with UBTF1/2 while silent NORs (indicated by the arrow) are devoid of UBTF1/2. Nuclei were visualized by DAPI. Supplementary Fig 2: UBTF1/2 regulates biological processes linked to chromatin and nucleosome assembly. (A, B) Gene ontology analysis using GREAT of UBTF1/2 ChIP-seq data. The most significantly enriched gene ontologies of UBTF1/2-bound genes for the 3705 peaks in NIH3T3 cells and the 9000 most significant peaks (based on p-value) in HMECs. (C) The most significantly enriched gene ontologies identified by GREAT of the common genes (Fig 1E) which overlap with UBTF1/2 ChIP-seq peaks within +/- 2 kb of their TSSs in the NIH3T3 and HMEC cell lines. (D) The most significantly enriched ontologies identified using GREAT of UBTF1/2 bound genes that overlapped with Pol II ChIP-seq peaks in NIH3T3 cells. The most significantly enriched ontologies identified using GREAT of the UBTF1/2 bound regions that are common between the HMEC and HMLER ChIP-seq data (E) and the UBTF1/2 bound regions that are unique to HMLER (F). Supplementary Fig 3: (A) qChIP analysis of Pol I (POLR1A) binding in NIH3T3 cells to ChIP-seq peaks as indicated. The % of DNA immunoprecipitated with anti- Pol I or rabbit serum (RS) antibodies was calculated relative to the unprecipitated input control. (n=5) Ave ± s.e.m, *p-value < 0.05 compared to corresponding RS samples. Amplicons at ETS and IGS of rDNA were used as a positive and negative control for Pol I binding, respectively. (B) IGV (Integrated Genome Viewer) screenshots of mapped reads from UBTF1/2 ChIP and input gDNA at HIST1 and HIST2 clusters in HMEC. (C) A list of the known number of canonical histone genes across the mouse and human genome as described in (Marzluff et al., 2002) and the number of canonical histone genes bound by UBTF1/2 in ChIP-seq data from NIH3T3 and HMEC. (D) Ubtf1/2 knockdown leads to cell cycle arrest. NIH3T3 cells transfected with sirEgfp, sirUbtf1/2#1 or sirUbtf1/2#2 for 48h were harvested and fixed in 90% ethanol. Cells were stained with propidium iodide at 50 µg/ml in PBS and analysed using the FACSCalibur and Cellquest Pro software (Becton Dickinson). The percentage of cells in G0/G1, S and G2/M phases were determined using Modfit 3.0 software. (n=3) Ave ± s.e.m, *p< 0.05. Supplementary Fig 4: UBTF1/2 regulates histone gene expression by mediating Pol II recruitment. (A) Total protein lysates of NIH3T3 cells transfected with sirEgfp or sirUbtf1/2#1 or sirUbtf1/2#2 for 48h and analysed by western blotting. (B) The results of (n=3) as A were quantitated by phosphoimager scanning, normalized to Tubulin levels and represented graphically, Ave ± s.e.m. **p-value < 0.01, *p-value < 0.05 compared to corresponding sirEgfp controls. (C) ChIP reChIP assay of the histone H2a, H4 and H1 genes in NIH3T3 cells using UBTF1/2 antibody for the first ChIP and antibodies to Pol I (POLR1A), Pol II or RS for the second ChIP. The % of DNA immunoprecipitated was calculated relative to the first ChIP. Error bars represent Ave ± s.e.m, (n=4), *p-value < 0.05. (D) Ubtf1/2 knockdown does not affect euchromatic and heterochromatic marks or nucleosome occupancy at histone genes. qChIP analysis of the histone H2a and H1 genes in sirEgfp or sirUbtf1/2#1 transfected NIH3T3 cells using antibodies against UBTF1/2, H3K27me3, H3K9me3, H4 hyperacetylation, H3K4me3, H3K9ac or total histones H3, H4 or H1. qChIPs were performed as described in Fig 4C, (n=3) Ave ± s.e.m, *P < 0.05 compared to sirEgfp ChIP. Supplementary Fig 5: The UBTF2 isoform mediates histone gene expression. (A) Tet-Off inducible RNAi resistant-rUbtf1, rUbtf2 and pRT (empty vector) 3T3-MEF cell lines were stimulated by doxocyclin for 5 days then transfected with sirEgfp or sirUbtf1/2#1 and harvested after 48 h for western blot analysis. (B) qChIP analysis of histone H2a, H1 and H4 genes using antibodies to FLAG in samples as in A (n=3) Ave ± s.e.m, *p-value < 0.05, **p-value < 0.01 compared to pRT/sirEgfp sample. qChIPs were performed as described in Fig 4C. Supplementary Fig 6: 48h after transfecting NIH3T3 cells with sirEgfp or sirUbtf1/2#1, IF for UBTF1/2 combined with FISH analysis for rDNA was performed. Nuclei were visualized by DAPI. Ubtf1/2 knockdown cells exhibit micronuclei (yellow arrows). Supplementary Table 1. List of genes belonging to the biological processes term (Chromatin assembly) identified by GREAT analysis to be significantly enriched with UBTF1/2-bound genes in NIH3T3 Genomic region-gene association table (76 regions, 85 genes) Gene region (distance to TSS) Asf1a region_320 (+657) Ctcf region_3371 (+651) Gm14483 region_3594 (-223684) Gm5382 region_3598 (+337549) Gm6026 region_3660 (-214579) Gm9998 region_900 (+480) H1f0 region_1282 (+415) H2afb1 region_1861 (-31271) H2afj region_3051 (-4053) H2afx region_3454 (+398) H2afy region_956 (+236) H2afy2 region_1262 (-191749), region_1261 (+232545) H3f3a region_214 (+547) H3f3b region_668 (+512) Hells region_1810 (+60118) Hist1h1a region_909 (-2641), region_910 (-1514) Hist1h1b region_871 (-2615), region_870 (+449) Hist1h1c region_902 (+277) Hist1h1d region_891 (-3550), region_892 (+314) Hist1h1e region_899 (+198) Hist1h2ab region_904 (-4749), region_905 (-4038), region_906 (+261) Hist1h2ac region_900 (-173) Hist1h2ae region_896 (-4868), region_895 (-3053), region_894 (-60) Hist1h2af region_886 (-2602), region_887 (+243) Hist1h2ag region_882 (-26) Hist1h2ah region_880 (-612), region_879 (+284) Hist1h2ai region_864 (+241) Hist1h2ak region_869 (-206) Hist1h2an region_872 (-122) Hist1h2ap region_873 (-71) Hist1h2ap region_876 (-205) Hist1h2bb region_903 (-1476), region_904 (-395), region_905 (+316) Hist1h2bc region_900 (-67) Hist1h2be region_899 (-1236), region_898 (+35400) Hist1h2bf region_896 (-1898), region_895 (-83) Hist1h2bg region_894 (-120) Hist1h2bh region_890 (-1284), region_889 (+187) Hist1h2bj region_881 (-2024), region_882 (-255) Hist1h2bk region_879 (-619), region_880 (+277) Hist1h2bl region_865 (-1805), region_864 (-520) Hist1h2bm region_865 (-4150), region_866 (-149) Hist1h2bn region_868 (-3788), region_869 (-90) Hist1h2bp region_871 (-4319), region_872 (-148) Hist1h2br region_874 (-1833), region_873 (-97) Hist1h2br region_875 (-1800), region_876 (+37), region_877 (+97428) Hist1h3a region_910 (+232) Hist1h3b region_906 (-1031), region_907 (+252) Hist1h3c region_905 (-1529), region_904 (-818), region_903 (+263) Hist1h3d region_894 (-3191), region_895 (-198), region_896 (+1617) Hist1h3e region_893 (+217) Hist1h3f region_889 (-887), region_890 (+584) Hist1h3g region_886 (-4109), region_887 (-1264), region_888 (+258) Hist1h3h region_864 (-965), region_865 (+320) Hist1h3i region_872 (-3943), region_871 (+228) Hist1h4a region_910 (-997), region_909 (+130) Hist1h4b region_907 (-4389), region_908 (+222) Hist1h4c region_901 (+215) Hist1h4d region_897 (+241), region_898 (+4122) Hist1h4f region_892 (-3703), region_891 (+161) Hist1h4h region_885 (-30980), region_886 (+265) Hist1h4i region_882 (-1623), region_881 (+146) Hist1h4j region_867 (+199) Hist1h4k region_869 (-3528), region_868 (+170) Hist1h4n region_873 (-1450), region_874 (+286) Hist1h4n region_876 (-1622), region_875 (+215) Hist2h2aa2 region_2262 (+883) Hist2h2ab region_2261 (+508) Hist2h2ac region_2261 (+420) Hist2h2bb region_2266 (-325) Hist2h2be region_2261 (-697) Hist2h3b region_2266 (+681) Hist2h3c1 region_2262 (-283) Hist2h3c1 region_2263 (+330), region_2264 (+1661) Hist2h4 region_2265 (+213), region_2264 (+16118) Hist4h4 region_3051
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